Jie:LabNotes/CpgSeq/2009-5-20
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bisulfite conversion of PGP samples
sample | sample concentration | sample volumn | ddH2O | conversion reagents | conversed DNA concentration and volumn | |
09_1 | NA21687*1 062507 | 241.1ng/ul x 1 tubes | 8ul | 12ul | 130ul | 95.4ng/ul x 10ul |
09_2 | NA21781*1 102307 | 240ng/ul x 1 tubes | 8ul | 12ul | 130ul | 120.2ng/ul x 10ul |
09_3 | NA21833*1 101807 | 253.9ng/ul x 1 tube | 8ul | 12ul | 130ul | 121.5ng/ul x 10ul |
09_4 | NA21660*1 053007 | 243.2ng/ul x 1 tube | 8ul | 12ul | 130ul | 120.3ng/ul x 10ul |
09_5 | NA21731*1 101807 | 217.4ng/ul x 1 tube | 10ul | 10ul | 130ul | 127.3ng/ul x 10ul |
09_6 | NA21846*1 100407 | 186.5ng/ul x 1 tube | 11ul | 9ul | 130ul | 151.3ng/ul x 10ul |
09_7 | NA21070*1 100406 | 329.7ng/ul x 1 tube | 6ul | 14ul | 130ul | 311.2ng/ul x 10ul |
Jurkat gDNA | 140ng/ul x 3 tube | 14ul | 6ul | 130ul | 255.6ng/ul x 20ul |
- Add 900ul ddH2O, 300ul M-Dilution Buffer and 50ul M-Dissolving Buffer to a tube of CT Conversion Reagent.
- Add 130ul of CT conversion reagent to samples.
- 98C 10min -> 64C 150min -> 4c overnight.
- Add 600ul M-Binding buffer to spin columns.
- Add the converted samples to the columns, close the gap and mix by inverting several times.
- Spin at 15,000rpm for 30sec.
- Add 100ul M-Wash Buffer, spin for 30 sec, discard flow-through.
- Add 200ul M-Desulphonation Buffer, wait for 20min, spin at 15,000 rpm for 30sec.
- Add 200ul M-Wash Buffer, spin for 30 sec.
- Place the columns into 1.5ml tubes, add 10ul M-Elution Buffer. Wait for 1min, spin at 15,000rpm for 30sec.
- Measure the DNA with Nanodrop:
RNA
set up the capture reaction
sample | sample concentration | 10xLigase buffer | template+cpg92k+H2O | template+cpg30k+H2O | reaction condition | template+v2.3(20ng/ul) vol+H2O | |
1 | Jurkat | 255.6ng/ul | 1ul | 1+4ul(#1_22.3ng/ul_05/20)+4ul | 55C anneal | ||
2 | Jurkat | 255.6ng/ul | 1ul | 1+4ul(#1_22.3ng/ul_05/20)+4ul | 60C anneal | ||
3 | Jurkat | 255.6ng/ul | 1ul | 1+5ul(#2_17ng/ul_05/20)+3ul | 55C anneal | ||
4 | Jurkat | 255.6ng/ul | 1ul | 1+5ul(#2_17ng/ul_05/20)+3ul | |||
5 | Jurkat | ||||||
6 | 09_1 | 95.4ng/ul | 1ul | 2+5ul(#2_17ng/ul_05/20)+2ul | |||
7 | 09_7 | 311.2ng/ul | 1ul | 2+5ul(#2_17ng/ul_05/20)+2ul | |||
8 | negative control | 0 | 1ul | 0+5ul(#2_17ng/ul_05/20)+3ul |
dNTP concentration 10mM(NEB) ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP: 0.58ul 0.1ul 0.1ul 0.2ul (10mM)0.02ul x300 174ul 30ul 30ul 60ul 6ul
95c 10min -> 55C 16h ->add 1ul SLN mix(2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP) -> 55C 4h ->
9 cycels (95C 1min -> 55C for 4h) -> 95C 1min -> 37C 1min ->
add 2ul Exonuclease I/III mix -> 37C 2h -> 94C 5min -> 4C hold.